NMDA-type glutamate receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the central nervous system, but are also implicated in many neurological and psychiatric disorders. NMDA receptors are composed of both glycine-binding GluN1 subunits and glutamate-binding GluN2 subunits. One GluN1 subunit has been cloned and there are four types of GluN2 subunits (GluN2A-D) with different developmental and regional expression levels that endow NMDA receptors with different functional properties. GluN1 glycine site agonists show rapid onset symptomatic relief in major depressive disorder, and reduction of NMDA receptor activity by inhibiting the activity of full endogenous agonists (e.g. glycine) hold considerable promise for the development of new antidepressant drugs. In addition, recent studies have shown that up to 20% of all patients with treatment-resistant childhood epilepsy disorders may have inherited or de novo mutations in the gene encoding the GluN2A NMDA receptor subunit. New partial GluN1 agonists with selectivity between GluN2 subunits have been designed, and a unique mechanism of action have been uncovered for a class of GluN2A-selective negative allosteric modulators (NAMs) that reduce affinity of glycine binding to GluN1. Using X-ray crystallography, site-directed mutagenesis, and electrophysiological recordings of NMDA receptor function, Aim 1 of this project will investigate the binding site of GluN2A-selective NAMs and Aim 2 will define binding contacts for new classes of partial agonists at the GluN1 glycine binding site. Aim 3 will examine the allosteric interaction between NAM and glycine binding sites in more detail using site-directed mutagenesis and engineered disulfide bonds. The relationship between allosteric inhibition by GluN2A- selective NAMs and GluN1 agonist efficacy will also be uncovered. This combination of aims will uncover new regions of NMDA receptors that can be exploited for engineering of subunit-selective ligands, and provide mechanistic and structural insight to enable rational design of novel, clinically-relevant modulators that reduce GluN1 glycine site activity.